Project Summary The conserved DNA mismatch repair (MMR) pathway repairs replication errors and reduces the mutational burden for organisms. In humans, MMR defects are found in some spontaneous cancers and in Lynch syndrome, also known as hereditary non-polyposis colon cancer and biallelic MMR deficiency syndrome, which are cancer predisposition syndromes. The fundamental unanswered question in eukaryotic MMR is how repair is targeted to the newly synthesized DNA strand. Strand discrimination is a crucial step in MMR, because repair mistakenly targeting the template strand would permanently fix the error in the genome. Accumulating evidence suggests that the Mlh1-Pms1 endonuclease, which is recruited to sites of error containing DNA by the mispair recognition factor Msh2-Msh6, plays a key role during eukaryotic strand discrimination. Furthermore, nucleotide-induced conformational changes in both complexes appear to play important roles during Mlh1-Pms1 recruitment and endonuclease activation, though the mechanism behind these events, as well as the role of Mlh1-Pms1 endonuclease activity during MMR, is unclear. However, one possible explanation is that recruitment and activation of Mlh1-Pms1 endonuclease activity function during strand discrimination to identify the newly synthesized DNA strand. Using newly developed techniques and building upon recent data, this proposal aims to examine Mlh1-Pms1 involvement during MMR and its potential role during strand discrimination by: (1) analyzing the role of nucleotide induced Msh2-Msh6 conformational changes during recruitment of Mlh1-Pms1 onto mispaired DNA (2) assessing the role of conformational changes in Mlh1-Pms1 for its recruitment and endonuclease activity, and (3) assessing interactions of Mlh1- Pms1 with pre-existing nicks in DNA to probe their possible role in strand discrimination. Mutant proteins and alleles will be utilized to examine the role of nucleotide-induced conformational changes in the Msh2-Msh6 and Mlh1-Pms1 complexes during recruitment and activation of Mlh1-Pms1 endonuclease activity using biochemical and genetic techniques previously utilized by the Kolodner laboratory. Mutant proteins will also be used to examine the role of Mlh1-Pms1 interaction with nicks and the role of Mlh1-Pms1 endonuclease activity using in vitro assays developed for this study. Completion of this proposal will make significant progress towards understanding the mechanistic role of Mlh1-Pms1 during MMR as well as identification of the eukaryotic strand discrimination signal.